RNA interference (RNAi) is a sequence-specific and post-transcriptional gene silencing process present in animals and plants and is mediated by 21-22 nt long RNA duplexes, called small interfering RNA (siRNA). This gene silencing mechanism appears to be particularly effective and holds great potential for decoding gene function and as gene-specific therapeutics. However, oligonucleotides do not diffuse freely across plasma membrane and imperiously depend on formulation within synthetic delivery systems.
RNA-splicing is a process by which a single pre-mRNA transcript can be alternatively spliced to produce multiple mRNA variants which in turn will be translated to different protein isoforms. In fact, up to 70% of human genes undergo alternative splicing and more importantly, up to 50% of human genetic diseases are known to arise from mutations that affect splicing. Moreover, aberrations in alternative splicing have been observed in many cancer-related genes. Therefore, optimization of drugs that can correct splicing mutations has recently become of great interest. Among ongoing splice correction trials, phosphorothioated oligonucleotides with 2′-O-methyl modifications are, particularly, found to be promising potential therapeutic agents for such diseases. Numerous studies have already reported on the therapeutic potential of splice-switching oligonucleotides by targeting several diseases caused by aberrant splicing such as Duchenne muscular dystrophy, β thalassemia and therosclerosis. While new generations of oligonucleotides are more resistant to degradation, their therapeutic use is still limited because of their poor pharmacological properties (Kurreck et al., 2003).
Non-viral delivery vehicles were initially developed for plasmid delivery (Felgner, 1999; Neu et al., 2005). They are generally cationic lipids or cationic polymers that interact electrostatically with the nucleic acid phosphate backbone to form stable complexes. These cationic complexes in turn bind to anionic proteoglycans present on cell surfaces, enter cells within membrane-coated vesicles and experience acidification on their road to degradative compartments. Escape from this pathway is required and relies on the incorporation of fusogenic lipids or endosomolytic functions within the complexes. Among the cationic polymers, polyethylenimine or PEI (patent application WO 96/02655) is certainly the most used plasmid DNA transfection agent because its high buffering capacity in the pH range between 5.0 and 7.5 facilitates rupture of endosomal membranes via a “proton sponge” mechanism. However, PEI, was shown to be a poor siRNA delivery agent (Grayson et al., 2006), especially in comparison to lipids.
Numerous approaches and hypotheses have been investigated to create efficient polymer-based delivery vehicles dedicated to siRNA. While siRNA duplexes and genes share a similar anionic charge density, the reduced number of anionic charges of a siRNA duplex in comparison to a plasmid DNA (average anionic charge of 7000) reduces the electrostatic cohesion of the soluble PEI with siRNA. Polyanionic proteoglycans present outside the cells and on the cell surfaces may then effectively displace PEI from the complexes, resulting in release of siRNA in the extracellular medium. No delivery and siRNA-mediated gene silencing can consequently occur.
Increased stability of oligonucleotide polyplexes may be performed by different means. siRNA duplexes could be artificially transformed into long structures, like plasmid DNA by equipment with self-complementary and overhanging nucleotides (Bolcato-Bellemin et al., 2007). Oligonucleotides could be conjugated with cholesterol for enhanced anchorage to cationic micelles (Zimmermann et al., 2006) or to cationic peptide so as to obtain an overall self-aggregating and cationic species (Fraley et al., 2006). Interestingly, it was shown that modification of PEI with natural amino-acids and in particular aromatic ones (patent application WO 2009/074970) led to a polymer with excellent siRNA delivery ability in eucaryotic cells. Use of aromatic alpha-aminoacids seemed particularly important since the amino-acid offers simultaneous possibilities to interact with the siRNA by electrostatic interactions (via the alpha-amine), hydrophobic and stacking interactions, leading to stable siRNA polyplexes.
However, nucleic acid translocation into the cell must involve a rupture of the lipid membrane integrity and some PEI conjugates may induce direct membrane destabilization. These polymers with membrane-perturbing activity at extracellular pH may exhibit hemolytic activity and lead to cell lethality. Thus, the use of cationic polymers, in particular PEI, often is limited by its cytotoxicity and so far has not been approved for use in humans.
To reduce the cytotoxicity of PEI, PEI has been modified, for example, with dextran sulfate, human serum albumin or polyethylene glycol, but all modified PEI show lower nucleic acid delivery efficiency than unmodified PEI.
Consequently, there is a strong need of developing cationic polymers that are efficient to deliver synthetic oligonucleotides and having a toxicological profile which is suitable for in vivo administration.